Transcribed by Erica Manion 7/25/2014

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Microbiology - Lecture 15 Chemotherapy of bacterial Infections by Dr. Tierno

[Slide 1] Chemotherapy of Bacterial Infections
[Dr. Tierno] Alright, its 11:00, I guess we could start.

[Slide #2] Biological Role of Antimicrobial Agents
Now there is an equation that is used in medicine. It is called the Theobald Smith Equation. It is
used to identify an infection. Sort of quasi mathematical. Theobald Smith came up with the idea
that infectious disease is directly related to the proportion of N times V divided by R. And the N is
the number of infecting organisms. The V deals virulence of those organisms, and the R is host
resistance to the infecting organism in addition to the host environment of the infection process. So
if you look at that equation, the numbers of organisms is very important. Thats called the
inoculum. For example, if you expose yourself to a foodstuff contaminated by Salmonella, you ingest
it, if there are not at least 10,000 cells that you ingest with that contaminated food, likely you will
not get food poising. It takes a minimum of about 10,000 cells or higher to give rise to a
gastrointestinal infection caused by Salmonella interitidis.
Whereas the Shigella anywhere from 10 to 100 cells would be sufficient to cause infection. And
theoretically an organism like a virus, a neural virus, may take just one single virion to cause
infection. So inoculum is important. And of course, so too is the virulence. Even though a Staph
aureus is very virulent because it has enzymes to attack your tissue and it is also able to produce
toxins. Other Staph, like Staph epidermidis on the skin dont have those enzymes. That doesnt mean
they wont cause infection in a person who is not immunocompetent, they can. Any organism has
the potential to cause infection. We are talking all things being equal, the definition of an infectious
disease, using the Theobald Smith equation, the R would be what the problem is in
immunocompetent, incompetent individuals, so they can be infected.
Now, using this whole concept of virulence in numbers,

[Slide #3] Clindamycin reduces the amount of glycocalyx in S. aureus and therefore
adherence ability
the virulence of an organism can be affected by antibiotics. Heres an example of Staph, with a
beautiful surface area rich in glycocalyx that adheres to tissue. Clindamycin is an antibiotic, even in
sub-lethal doses, interferes with protein synthesis and this glycocalyx is not produced strong
enough to be able to overcome host response. In fact, it wont adhere. Remember, we said the first
thing an organism has to do is come to the host. Meets the host. In this case, you can affect the
attachment by sub-lethal doses of an antibiotic called clindamycin, which we will talk about.

[Slide #4] Table: Effect of clindamycin on growth and production of toxic shock syndrome
exotoxin of Staphylococcus aureus
Heres an example. I talked about of toxic shock syndrome. If a patient comes in to a hospital with a
diagnosis of toxic shock syndrome, the most important thing you could do, rather than
administering an antibiotic that affects the cell wall and you know what happens when you affect
the cell wall, the organism blows up. In so doing, it will release whatever toxins is being
manufactured by that cell en mass, into the patient, and may be deleterious to the patient. Here is
an example where clindamycin, in sub-lethal doses, in other words, 1/100 of a microgram, which is
a millionth of a gram per mL, actually doesnt stop the growth of the organism, but will suppress
toxin production. So that can be a lifesaver in a case like that. So, heres where you interfere with
toxin production. Of course, you can also interfere with the growth of an organism by just
administering an antibiotic. They can kill them off or suppress the growth so your immune
response has an ability to come in to play and defeat the organism.

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[Slide #5] History
Now, if you go the history, history is so overwhelming. I know you dont have spare time, but it is
really interesting to read ancient descriptions and texts. Going back to the ancient Chinese,
Egyptians, ancient Indians, Mesopotamians, etc. They didnt know why, but once they took
fermented rot from trees and plants and put it on a wound of a soldier, the soldier healed. Without
that, the soldier would die. They empirically learned about how to apply certain natural things to
wounds. It turned out, it isnt just antibiotics that are produced, it is a competition of organism with
pathogens. Theres more than just, you can say well the fungus is producing antibiotics.

It may produce antibiotic substances, but it is also the competition that occurs. It wasnt until the
17
th
century that the use of quinine for malaria was the first antimicrobial that was specific for a
particular disease.
In 1847 one of the most important discoveries was made by Ignas Semmelweis. He was an
obstetrician, and he noted in the days when the obstetrician would go from one woman to deliver,
another woman to deliver, and wipe their hands on their coats, go to the next. They knew nothing
about germs and microorganisms passing and inoculating one woman to another. They died of
childbed fever. If only one woman had that Strep piogenes infection, their hands were
contaminated. They would wipe them on their coat and go to the next woman to deliver a baby. So
people in those days knew, you go to the hospital to deliver a baby you could die! So they stayed at
home to deliver a baby, richer people did. Poorer people, [inaudible], they were in a hospital, and
they died en masse. Ignas Semmelweis developed chlorinated lime that he would wash his hands
with in between patients. He didnt know why, he said it rids the miasmis[?] that are on hands, so
that was useful. Of course it was a long story. He was challenged by some people who said that is
baloney. You get the same death with your method that you do with my method, which is no
method, just wiping. And what he did is, he did not make these people wash their hands with the
chlorinated lime, and they claimed they did and they got the same death. He actually went insane
for a while because he knew he was right. And in fact he died because in the end of his life he was in
a hospital doing autopsies on patients and he pricked himself with a scalpel and he actually got one
of the women who had child bed fever, inoculated, with that scalpel, his hand, and he died of Strep
piogenese, massive infection. Its an interesting story.
Pasteur observed the Anthrax bacilli were actually killed if you spray in throats, if you had oral
anthrax, another organism. Various types of organisms. He used, its not important to know which,
they used soil bacteria, so to speak. And he was the first to observe an antibiotic effect and report
it.
And Paul Ehrlich gets the credit with being the father of chemotherapy by developing an arsenical
compound. The 606
th
permutation of an arsenical compound that kills syphillis. And many of the
women of the evening went to him and were eager to participate in studies to kill the dread disease
that they had. And unfortunately for 605 trials, those women died of arsenic poisoning. But his
606
th
permutation displayed selective toxicity. He gets the credit because of selective toxicity,
which we will touch base on, and he is the father of chemotherapy.
Then of course, in 1929 Fleming, by chance, discovered penicillin. But it wasnt used until WWII. It
was mass produced by Chain and Florey. And in 1935, prior to penicillin being used, sulfonamides
were identified by Domagk and used to treat. Sulfa drugs were long known to treat various types of
infections. And Waksman in 44 gets credit for streptomycin, and he was the one who coined the
term antibiotic, 1944. And from then on, pharmaceutical industry took over, and we have a plethora
of antibiotics. Unfortunately, we are running out of antibiotics because of resistance problems,
which I will discuss in another lecture.

[Slide #6] Paul Ehrlich
Here he is, Paul Ehrlich
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[Slide #7] No title, starts with The single essential attribute of a clinically .
The principle of selective toxicity, if you give somebody a drug like arsenic, the idea is to kill the
organism, not the patient. 605 times it killed the patient, so the selective toxicity of that drug was
nill, zero. But here, you want to make sure, and in bacterial cells there is no counterpart, usually, in
the normal human host cells. They have different ribosome system, and they have cell walls, we
dont, so there are many facets of the bacteria that can be utilized to produce drugs will not hurt us.
But if you take a drug for a long enough period of time, even though you have no site, there are
other things it can do to you. So the toxicity causes damage like inactivating, or killing or interfering
with target cells is the whole point that you want selectivity. You want to kill the bug, not the
person.

[Slide #8] Chemotherapeutic index
There is an index, you can reverse this, you can put the concentration producing host damage on
top, and the bottom, so therefore you get a different number, you get a whole number, a fraction,
doesnt matter. The whole concept is the same. The drug concentration producing the specific
effect versus the drug concentration that would produce host damage. That is what the
chemotherapeutic index is, and they figured that out for every drug. Theres a certain dose you
need, and theres a certain dose if you exceed, you may hurt a patient. For ex, aminoglycosides are
very useful drugs, we will touch on this. They can kill organisms very nicely. However, if you go
beyond a certain level, they may not kill you, but they can cause ototoxicity - loss of hearing,
nephrotoxicity, and other drugs cause other problems in a person. So it is very important to pay
attention to chemotherapeutic effect.

[Slide #9] No title, figure of bacteria
Now this slide gives you the ways that antibiotics interfere with bacteria. Its important for you to
keep abreast of this and understand it. Most drugs affect the cell wall. Most drugs. The drugs that
you are familiar with like penicillins, cephalosporins, vancomycin, and drugs you use on your finger
like bacitracin and the like are cell wall interferers.
Second would be a protein synthesis, thats the second category. The drugs like erythromycin,
chloramphenicol, and clindamycin interfere with the 50S subunit. Therefore interfere with proteins
being produced. Remember what I said about toxic shock and clindamycin. Clindamycin is a very
good drug for that. Now, there is also 30S inhibitors. They interfere with production of protein on
the 30S ribosomal unit. There is tetracycline, streptomycin and the aminoglycosides. These three
are aminoglycosides, gentamicin, tobramycin, and amikacin. Well talk about them.
So the next popular would be the paraminobenzoic acid interference of sulfonamides and
trimethoprim. They interfere with folic acid metabolism. Many of you have had drugs like Septra or
Bactrim, which are sulfatrimethoprim combinations, to treat urinary tract infections and other
types of infections. That is common. So that is folic acid metabolism.
Then there is DNA and RNA interference. Either an RNA polymerase like rifampin. A DNA
dependent polymerase enzyme to interfere with mRNA. And DNA replication, you can have the
quinolones like ciprofloxacin and other similar drugs, moxifloxacin, that interfere with DNA gyrase
enzyme and therefore DNA synthesis.
The last category is cell membrane active of which there is one, the polymyxins. What it does, it is
more like a detergent. This particular drug gets in between the cell wall and the membrane, opening
it so that things go in and out of a cell. When a cell loses selective characteristics of allowing things
to come in and out of a cell, it dies.

Generally speaking, any antibiotic that interferes with cell wall production, would it be a lytic drug
or would it be, in other words cidal, or would it be a static drug? Lytic, it would blow up the cell.
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Remember what I said, by definition.

[Slide #10] Need to Know
So what you need to know, touching on that, are these five things about every category of antibiotic.
I make it simple, because I make a chart that displays these five things for every antibiotic. What
type of antibiotic is it? For example, penicillin cephalosporins are Beta lactams.
The site of action of a drug, is it cell wall, protein synthesis or what? Of the five things I talked about.
Is it a cidal drug or static drug?
Does it effect broad spectrum, meaning both gram negatives and gram positives like E. coli and
Staph aureus, or is it selective for gram positives like Staph aureus only and Strep only, the gram
positives, or the gram negatives like E. coli only.
Is it, whatever other little tid-bit, one tid-bit of pertinent information? Is it perenteral, meaning you
cant take it by mouth, its gotta be administered intramuscularly or intravenously, not by mouth,
which is PO. Is it affected by food? Like certain drugs like tetracyclines might be affected by food.
Thats the kind of stuff you need to know.

[Slide #11] Image, gram pos vs gram neg + drug molecules
Now looking at this chart, on the left is gram positive bacteria, on the right are gram negatives.
Look at the cell envelope of each. Gram positives allow free flowing drug. Drug molecules can easily
come into a gram positive cell and reach, the lets say, the penicillin binding proteins which are on
the inside of the bacterial membrane, and are called transpeptidase.
Whereas the gram negative is different. The gram negative trilaminar structure requires the
antibiotic to come through a porin, a pore in the membrane. And that pore allows aqueous
molecules to pass through, therefore it is more restrictive. So very large antibiotics by definition
would not get in.
Here you have hydrophilic character, water molecules and others, easy delivery, 600 800 to 1000
daltons in size, and it can vary up to 6000 daltons in size can pass through the pore.

[Slide #12] Peptidoglycan synthesis
Lets look at the following antibiotics that interfere with the cell wall synthesis, in this case,
peptidoglycan synthesis. This particular drug, cycloserine, is not used a great deal. But never the
less it interferes with the first stage of cytoplasm. At the very basic level, if you look at L alanine to D
alanine, D-alanine to double D-alanine, you see there are two enzymes, the alanine racemase and
synthetase that are important to bring that L-ala to D-alanyl-D-alanine. And that is required in the
first step of peptidoglycan synthesis. Well, cycloserine is a mimic, it is an analog. It looks just like
the D-alanine, and Ill show you a picture later. It adheres to the enzymes, both of them, better than
either alanine or L-alanine adheres. So therefore it interferes at a greater preponderance, at a
greater level than that which gets through. Now any antibiotic is not 100% immediate. It is over
time that it occurs.

[Slide #13] Figure 10 14
Here is the analog. If you look at the right hand side, they look almost identical. Not the left hand
side, the right hand side is the active side, and you can see how that would adhere to the enzyme.
The enzyme would think cycloserine is D-alanine.

[Slide #14] Cycloserine
Cycloserine. What I said, the five things you will need to know, structural analogue of D-alanine,
site of action: first stage of cell wall synthesis, low molecular weight precursors. And is it cidal or
static? Anything that deals with cell wall interference is cidal. Does it effect gram positive and gram
negative? Yes. But it is also used for TB mostly, nowadays. It is broad spectrum. Other, central
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nervous system toxicity. Thats why it is restricted to TB.

[Slide #15] No title, starts with 2
nd
stage cw synthesis
Now, a second drug that interferes with cell wall synthesis is bacitracin, which we no longer use
internally. We use it on cuts and scrapes. It can be a uh, drug that causes problems in people. But
here it interferes with the second stage. It prevents the dephosphorylation of the lipid
pyrophosphate. Pyrophosphate is on the left, bacitracin actually blocks by binding to this
pyrophosphate, preventing the movement to a single phosphate lipid. Its a very simple thing, but

[Slide #16] Fig. 9 - 14
look at the size of it. It is a pretty big. What would that tell you, primarily? What bugs would this
be able to get into? Gram positive, not gram negatives. And its mostly - and it is a polypeptide drug.
You see all the amino acids? Polypeptide drug. That is the class.

[Slide #17] - Bacitracin
So again, its polypeptide, its second stage of cell wall activity. Inhibits dephosphorylation of the
lipid pyrophosphate. It is cidal because it interferes with cell wall. But the spectrum is usually gram
positive, like staph, because of its size. But it can give systemic toxicity, thats used only topically.
Thats an important bit of information.

[Slide #18] Below is an example of a G+ bacterial cross bridge
I made it as easy as possible to just know groups of these things and examples. Youll get your
pharmaceutical chemistry later. Youll get your treatment modalities later. Right now look at the
concept. Heres an example of how penicillin acts. It takes Ordinarily there is an enzyme celled
transpeptidase that takes this last glycine residue and attaches to the penultimate (in other words
second from the top) D-alanine molecule and binds them for a cross bridge. In gram negatives its a
different process as I mentioned up there but lets look at this process.

[Slide #19] Figure 3.9: Penicillin
Now uh, why is it so easy for the cell to think penicillin is D-alanine, D-alanyl-D-alanine? Why?
Similar. Analog. Its an analog. Even though it is a beta lactam antibiotic, which is the category I
want you to say, but it is an analogue of this! Very simple. Manufacturers are going crazy trying to
develop things like this, analogs, that interfere with enzyme reactions of organisms, and theyve
been very successful. Unfortunately, organisms have developed resistance.

[Slide #20] Beta lactams: Penicillins and Cephalosporins
So very simply put, beta lactams, 3
rd
stage of cell wall, covalently bond. Covalent is not moveable.
The bond is firm so it doesnt come apart. So they covalently bond to the enzyme PB, penicillin
binding protein, thats what that means, Preventing the crosslink, which, between the glycine
residues on the penultimate, as opposed to ultimate, which is the top. It is cidal, broad spectrum,
depending upon the drug you use. Many cephalosporins go both ways, penicillins go both ways, to
different kinds of gram negatives, like not necessarily E. coli in all cases. It could be broad spectrum
by killing Haemophilus, which is another gram negative. Oral and parenteral, that is the usefulness
of this drug. And it has the least amount of side effects, penicillin drugs. But remember, use
anything long enough, it can interfere with things.

[Slide #21] Figure 11-3 Basic structures of B-lactam antibiotics
And this is a compilation of the different types of beta lactams. Penicillin with the thiazolidine ring.
Its that five sided ring, as opposed to the dihydrothiazine, which is six sided, of the cephalosporins.
Very similar to beta lactam portion is the same on all of these.
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[Slide #22] - Vancomycin
Now, vancomycin also interferes with the cell wall. Interestingly, its a very large molecule. So we
know right off the bat, whats the spectrum? Gram positive.

[Slide #23] No title. Flow chart showing vancomycin activity
This is an interesting drug. Originally, we thought that vancomycin works only here, taking
petidoglycan, using a transglycolase (I think he means transglycosylase?) enzyme, making it a more
complex peptidoglycan, linking up with the pentapeptide, uh, glusyl (? 1:05:45 on iTunes), uh,
chemistry. And now, we realize that it is not only active with the transglycolase reaction, but it
actually is also it can interfere with transpeptidase. So it does both enzymes. We didnt appreciate
it. With a greater frequency with transglycolase, current research is showing it interferes also with
the transpeptidase enzyme. Its picked up just like beta lactams are.

[Slide #24] Key steps in bacterial cell wall synthesis figure
If fact, here is the reaction. The key steps in the cell wall synthesis are here, and vancomycin
interferes with the transglycosylase enzyme, as well as the transpeptidase enzyme. There is a
moiety on that big molecule that seems to be at play and is not always in proper order to link on to
the transpeptidase enzyme. Look how big this is. But it is always able to link onto the
transglycosylase enzyme. So it does both, both enzymes. So far, its not magic. These antibiotics
interfere with normal development of the cell wall.

[Slide #25] - Vancomycin
So, this is a glycopeptide, in that there is glucose base molecules with peptides as you see there, big
molecule. Site of action, we already discussed, binds to the D-alanyl-D-alanine precursors. Cidal.
Gram positive spectrum because of its size. It is parenteral in most cases if you have a systemic
infection. However, because its not absorbed orally, however for C. difficile, Clostridium difficile,
gastroenteritis, you take it orally. Because it does not get absorbed but that is where you want the
action. In the gut. And it works, so does a couple of other antibiotics for C. difficile. Remember what
I said, in the end, when all else fails, they actually replace the flora by insertion of a normal persons
stool, with the proper uh, firmicutes versus bacteroidetes relationship, into the rectum. And you get
cured in most cases, greater than 90% cure. If not you keep trying. And the prescription for
Clostridium difficile is thats thats where it is mostly used, it was. Now they restrict that and use
flagyl instead, which is not as good. And of course, this can also be used for MRSA. Methicillin-
resistant Staphylococcus aureus.
Even that has been shown to be a problem because there have been intermediate susceptibility to
vancomycin by MRSA strains. And the reason for this quasi resistance, and sometimes resistance,
comes from its overuse against Staph and overuse for C. difficile. We are now having vancomycin
resistant enterococci, which are part of the normal gut flora of people, and that can cause infection
because it is not eradicated.

[Slide #26] - AMINOGLYCOSIDES
Now, there is another group of antibiotics. Those were the cell wall active agents that we are going
to discuss. The second group we are going to discuss have to do with protein synthesis. And this is a
very interesting group. The aminoglycosides. It means you have amine groups and what looks like
glucose residues there, modified. There are 4 aminoglycosides represented here. Kanamycin was
one of the first aminoglycosides. It can be a dangerous drug. It was used a lot on patients who had
no kidney function. Because of that, we figured maybe its not going to raise havoc in these patients.
But even correct dosing in these anuric patients, many of them developed ototoxicity and lost their
hearing. So these drugs are very potent drugs. So, theres gentamycin, tobramycin, kanamycin, and
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amikacin. Those are the four drugs. [responding to some students leaving] I hope they are catching
a bus! haha

[Slide #27] Inhibition of the 30S ribosome-RNA complex formation
Now here is where they interfere in two ways with the proteins. You can have the 30S, these are
30S active, they are active at the 30S site. The aminoglycoside, thats AGLY, thats what that means,
is irreversibly bound to that ribosome, making the ribosome actually shut down protein synthesis.
Simple. Just latches on to the ribosome. And, you can also have, sometimes the ribosome is active
and produces a protein. However, the protein is a nonsense protein. A protein that is not useful to
the bacterium. So even if it produces a protein it is going to misread the code on the messenger
RNA, and will therefore be defective and of no use to the organism. An interesting group of
antibiotics.

[Slide #28] Figure 10-19
There is an anomaly that occurs. We noted on certain cultures in the laboratory when you get an
organism and you prepare an antibiotic susceptibility pattern to it - you challenge the microbe with
these discs, or you did it in another way, and well talk about it in a lab section. Usually you get that
lab section before you get this lecture. We noticed that in the presence of the aminoglycoside, the
organism grew. In the presence of the aminoglycoside. If you take the aminoglycoside away, the
organism dies. We found that some organisms develop a tendency to be dependent for their
growth, on the aminoglycoside. So if a clinician has a patient on aminoglycoside, he notes or she
notes that that patient will defervesce, appear to get better, all of a sudden rebound, getting worse,
still on the drug. We check the organism, it grows only in the presence of the antibiotic. We tell the
clinician, take the drug away. Take the drug away, the organism dies. It has developed dependency
on an antibiotic. Things like that can occur, and thats what this shows. You can see how. Youre
recognizing the region on the messenger RNA and yet, because of the presence (in this case) of
streptomycin, it warps the reading. There is a misreading caused by the streptomycin. So an
organism develops a reactivation ability by correcting that misreading, correcting it, so that its not
interfered with by the streptomycin. You see it? Inactive dependent ribosome. Reactivated
ribosome. So in some cases, a drug can actually foster the growth after a while. Remember
organisms mutate. Theyve been around for approximately 4 million years. Their chemistry is
amazing. And by the way, its the same chemistry of all plants and animals. There are exceptions,
and including humans. We use DNA, they use DNA, RNA, ribosomes. Its a very similar chemistry.
Alright so thats an important thing to know, this distortion effect. So if you take away the drug, the
bug will die.

[Slide #29] Aminoglycosides (gentamicin, tobramycin, amikacin, neomycin, streptomycin)
These aminoglycosides as a class irreversibly bind to the 30S ribosomal subunit and they interfere
with protein synthesis either by completely shutting it down or by misreading. Of course, um, here
you have irreversible binding, you have cidality. Spectrum is gram positive and gram negative but
not anaerobes. It does no good for anaerobic bacteria. And it can only be used parenterally. You
need to get IV or IM. Which is a problem, and that is why it is used mostly in hospitals, because it is
poorly absorbed orally. You dont get much of a dose if you take it by mouth. And they penetrate
the CNS poorly. Youd have to inject the spine if you want to get it in. And they are nephrotoxic and
ototoxic. And here is a very important point. They are synergistic. Synergy is enhancement.
Antagonism is the opposite of synergy. If you give two drugs and they have an enhanced effect,
greater than the sum of the two, that is synergy. If you have two drugs that are antagonistic, you
have less than the best drug, it would be less than the effect, the one drug. So you want synergy.
These drugs are synergistic with all beta lactam antibiotics and are used together. Used [?]
cephalosporins or penicillins plus aminoglycosides in serious infections. In fact, there is a
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facilitation of uptake of the aminoglycosides. These are tough molecules to take in, and this actually
assists it, and maybe thats the real basis of the synergistic effect.

[Slide #30] Tetracyclines
Im not going to start the tetracyclines. This is a good time to stop. If I start the tetracyclines well
go to 12:00 and I know you people have something, you say. Although, people freely walk out and
freely come in while Im talking. But the next group of lectures, I think next week, Tuesday and
Thursday I have you, Ill see you then. Ok? Ciao.